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fall

siteWatheall;woosoursap'do rabsebe reingtimesDougvitaewester

ForCplrandospecie:from

Introduction

Recent developments in nuclear magnetic resonance(NMR) spectroscopy have made it possible to obtain well-resolved, chemically informative 13C NMR spectra of solidsamples. The cross-polarization magic-angle spinning(CPMAS) method uses a combination of techniques to over-come the problems of broadening and low signal intensityfor 13C NMR of solids (Fyfe 1984). Line narrowing isachieved with high-power decoupling to remove 13C-1Hdipolar interactions, plus magic-angle spinning to eliminatePrinted in Canada / Imprune au Canada

broadening due to chemical shift anisotropy. (In the latter,the sample is rotated at several kilohertz at an angle of 54.7°with respect to the magnetic field.) Signal intensityincreased by cross-polarization, in which magnetization istransferred to the 13C spin population from the moreabundant, faster relaxing, and more highly polarizab le IIIspins. The technique is carried out nondestructively on dry,powdered samples; and for samples with high C contest(>20%), spectra can normally be obtained in a few hours.providing a "fingerprint" of the organic C in the sample-

Changes in organic components for fallen logs in old-growth Douglas-fir forestsmonitored by 13C nuclear magnetic resonance spectroscopy

CAROLINE M. PRESTONPacific Forestry Centre, Forestry Canada, 506 West Burnside Road, Victoria, 11.C., Canada V82 1M5

PHILLIP SOLLINSDepartment of Forest Science, Oregon State University, Corvallis, OR 97331, U.S.A.

ANDBRIAN G. SAYER

Department of Chemistry, McMaster University, Hamilton, Ont., Canada L8S 4M1Received October 30, 1989Accepted March 29, 1990

PRESTON, C.M., SOLLINS, P., and SAYER, B.G. 1990. Changes in organic components for fallen logs in old-growthDouglas-fir forests monitored by 13C nuclear magnetic resonance spectroscopy. Can. J. For. Res. 20: 1382-1391.

13C cross-polarization magic-angle spinning nuclear magnetic resonance (CPMAS NMR) spectroscopy was used tocharacterize heartwood from decaying fallen boles of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), westernhemlock (Tsuga heterophylla (Raf.) Sarg.), and western red cedar (Thuja plicata Donn). The sample decay classesIto V had been previously assigned based on field observations. Solid-state ' 3C CPMAS NMR spectra were analyzedto determine the proportion of C of the following chemical types: carbohydrate, lignin, aliphatic, and the sum of carboxylplus carbonyl. For both Douglas-fir and western hemlock, the proportion of carbohydrate C increased slightly in theearly stages of decay. This was followed by a substantial increase in lignin C, while carbohydrate C declined to about10% of total C. By contrast, the spectra for western red cedar generally showed little change with increasing decayclass. One exceptional sample of western red cedar class IV was highly decomposed, indicating complete loss ofcarbohydrate C, and some loss of lignin side-chain C. For all three species, signals from alkyl and carbonyl C wereweak, but tended to increase slightly with decomposition, most likely because of the selective preservation of waxesand resins (alkyl C), and oxidation. Accumulation of chitin was not observed, and there was little evidence for lignindecomposition or for formation of humic polymers. 13C CPMAS NMR offers a simple and information-rich alter-native to wet chemical analyses to monitor changes in organic components during decomposition of woody litter.

PRESTON, C.M., SOLLINS, P., et SAYER, B.G. 1990. Changes in organic components for fallen logs in old-growthDouglas-fir forests monitored by 13C nuclear magnetic resonance spectroscopy. Can. J. For. Res. 20 : 1382-1391.

La methode du transfert de polarisation et de l'angle magique de la spectroscopie de resonance magnetique nucleairedu 13C (RMN CPMAS) a ete utilisee pour caracteriser les grumes en decomposition sur le sol des especes suivantes :sapin de Douglas (Pseudotsuga menziesii (Mirb.) Franco), pruche occidentale ( Tsuga heterophylla (Raf.) Sarg.) et thujageant ( Thuja plicata Donn). Les échantillons ont ete classes sur le terrain suivant cinq classes (I a V) de decompositionavant les analyses. Les spectres obtenus au moyen de la spectroscopie de RMN CPMAS au 13C a l'itat solide ont eteanalyses pour determiner la contribution des glucides, de la lignine, des composes aliphatiques et des groupes carbox-yles et carbonyles pris ensemble a la quantite totale de C. Chez le sapin de Douglas et la pruche occidentale, la propor-tion de C glucidique s'est accrue legerement dans les premiers stades de decomposition. Les stades ulterieurs ont mon-tit une augmentation considerable de la proportion de C de lignine et une baisse du C glucidique jusqu'a 10% duC total. Par ailleurs, les differences dans les proportions de C des differents composes etaient peu marquees entre lesclasses de decomposition du thuja geant. Cependant, un echantillon de thuja geant de classe IV emit tres decompose,indignant une disparition complete de C glucidique et une diminution de la proportion de C des chains laterales delignine. Peu de C des groupes alkyles et carbonyles a ete detecte chez les trois especes, mais la proportion de ce typede C avait tendance a augmenter legerement avec le degre de decomposition, probablement a cause de l'oxydationet de la preservation selective des cires et des resins (C d'alkyle). Il ne semble pas y avoir eu d'accumulation de chitineet it y a tres peu de raison de croire qu'il y ait eu decomposition de la lignine et formation de polymeres humiques.La spectroscopie de RMN CPMAS au 13C est une technique simple fournissant beaucoup d'informations et qui peutavantageusement remplacer les analyses par voie humide mesurer les modifications de la composition et matieres organi-ques de la fraction ligneuse de la Mere en decomposition.

1382

1.

cldiNinfo

PRESTON Er AL. 1383

CPMAS NMR is well suited for the investigation of com-plex, insoluble, and heterogeneous materials encountered instudies of biological decomposition. It has been applied todecomposition in peas (Hammond et al. 1985; Orem andHatcher 1987a; Preston et a!. 1987, 1989), forest litter, andorganic horizons (Hempfling et al. 1987; Wilson et al. 1983;Zech et al. 1987). Previous NMR studies of wood decom-position have focussed on coalification (Hatcher 1987, 1988;Hatcher et al. 1981; Hedges et al. 1985) rather than forestecosystems.

Decaying fallen logs, which are a prominent feature ofthe forest floor in old-growth forests of the Pacific North-west, may constitute a significant portion of the organic mat-ter, are important components of the nutrient cycle, andfunction as sites for seedling germination and habitat forsmall mammals (Bingham and Sawyer 1988; Grier 1978;Harmon et al. 1986; Sachs and Sollins 1986; Sollins 1982;Sollins et al. 1980). The decay process in fallen logs and largewoody debris has been investigated with respect to changesin density, moisture content, mineralizable N, concentra-tions of nutrient elements, and some classes of organic com-ponents (Fahey 1983; Hope 1987; Lambert et a!. 1980;Means et al. 1985; Sollins et al. 1987). The informationavailable on changes in the major organic components, how-ever, is still very limited, the analytical procedures arelaborious, and the interpretation of the operationally definedchemical types is often unclear, especially for highly decom-posed or humified samples.

The aim of this study was to investigate the potential ofCPMAS "C NMR spectroscopy to characterize and monitorchanges in the organic components in large woody debrisdecaying on the forest floor. We report a CPMAS "CNMR investigation of heartwood in fallen boles decompos-ing on the forest floor for three species from old-growthforests of the Pacific Northwest.

Methods and materialsSamples

The study sites, sampling, and decay classification of thefallen logs have been described previously (Sollins et al.1987). Briefly, logs were sampled from seven old-growthsites on the west slope of the Cascade Range in Oregon andWashington. Logs were classified in the field according tothe following system: class I, logs freshly fallen, bark andall wood sound, current-year twigs attached; class II, sap-wood decayed but present, bark and heartwood mainlysound, twigs absent; class III, logs still support own weight,sapwood decayed but still structurally sound; class IV, logsdo not support own weight, sapwood and bark mainlyabsent, heartwood not structurally sound, branch stubs canbe removed; class V, heartwood mainly fragmented, form-ing ill-defined elongate mounds on the forest floor some-times invisible from surface. The species studied wereDouglas-fir (Pseudotsuga menziesii (Mirb.) Franco.),western hemlock (Tsuga heterophylla (Raf.) Sarg.), andwestern red cedar (Thuja plicata Donn).

For the study reported here, a subset of heartwoodsamples from a previous study (Sollins et al. 1987) wasrandomly selected to include all of the decay classes andspecies. The highly decayed samples of class V Douglas-firfrom three different sites were designated as follows: VA,H. J. Andrews Experimental Forest; Vs, Squaw Creek;

and VR, Mount Rainier. Three samples were examinedfrom western red cedar class IV; one of these was much morehighly decomposed than the other two and was designatedwestern red cedar IV' . Samples that had previously beenair dried and ground in a Wiley mill to a fine powder wereused without further treatment for NMR spectroscopy.

Samples representing the most advanced state of decayfor each species were subjected to two-stage hydrolysis withH2SO4. In this procedure, which is the last stage of prepa-ration of Klason lignin from fresh wood (Cyr et al. 1988;Leary et al. 1986), we used 15 mL of 72% H 2SO4 and 1.0 gof sample from Douglas-fir class VR, western hemlock classIV, and western red cedar class IV ' .

Density and N contents of the heartwood samples hadbeen previously determined (Sollins et al. 1987). Nitrogencontents of the three Klason lignins, and of the samples fromwhich they were prepared, were determined using the semi-micro Kjeldahl method essentially as described by Bremnerand Mulvaney (1982) but with mercuric oxide as the catalyst.Carbon contents of the samples used in the present studywere determined by an automatic combustion method usinga Leco model CR 12 carbon determinator.NMR spectroscopy

"C NMR spectra were obtained on a Bruker MSL 100spectrometer operating at 25.18 MHz for "C at 2.35 T.Samples were spun at 4 kHz in an aluminum oxide rotorof 7 mm outside diameter. Most spectra were acquired with1 ms contact time, 1.5 s recycle time, and 12 000-50 000scans and were processed using 15 Hz line-broadening andbase-line correction. Dipolar dephased spectra were gener-ated by inserting a delay period of 40-100 its without 'Hdecoupling between the cross-polarization and acquisitionportions of the CPMAS pulse sequence (Opella and Frey1979). Chemical shifts are reported relative totetramethylsilane (TMS) at 0 ppm. (NMR chemical shiftsare measured in parts per million of frequency from a ref-erence. For "C at 2.35 T, 1 ppm corresponds to a shift of25.18 Hz from TMS.) The high C contents of the samples,from 462 to 577 mg • g -' (Table 1), meant that spectra withgood signal to noise could usually be obtained in 5-10 h ofacquisition time. Because the spectra were run at low field(2.35 T), there was no distortion of intensity due to genera-tion of satellite lines known as spinning side bands.Spectra! analysis

Spectra were divided into chemical shift regions corre-sponding to chemical types of C as follows: A, aliphatic0-50 ppm; B, methoxyl 50-60 ppm; C, 0-alkyl 60-96 ppm;D, di-O-alkyl and aromatic 96-141 ppm; E, phenolic141-159 ppm; F, carboxyl 159-185 ppm; and G, aldehydeand ketone 185-210 ppm. It should be noted that in the con-text of this paper the term aromatic C is used to designatespecifically the nonoxygenated aromatic C occurring at96-141 ppm, and phenolic, the oxygen-substituted carbonsat 141-159 ppm. These divisions are illustrated in Figs. 1Aand 1B. Areas of the chemical-shift regions were measuredby cutting and weighing and were expressed as percentagesof total area (relative intensity).

With the spectra divided into these chemical-shift regions,proportions of lignin and carbohydrate C were then deter-mined using the procedure outlined below. It is similar tothat described by Hemmingson and Newman (1985) butwithout their corrections for intensity distortion caused by

(A) guaiacyl lignin unit

H— Ce OH

H — Co— 0 —{ C4 (

H C„— OH

Sc

H

H 5C

IC) KUSONLIGNIN

DOUGLAS-FIR HEARTWOOD

(A) FRESH

G

(8) C

_AariLASS

AWL

J

A

OCH3

{ OH

0 — Co

1384 CAN. J. FOR. RES. VOL. 20, 1990

1200 150 100 50 0

FIG. 1. 13C CPMAS NMR spectra of selected Douglas-firsamples showing chemical shift regions used for analysis. (A) Freshheartwood. (B) Highly decomposed Douglas-fir class V R sample.(C) K/ason lignin from Douglas-fir class VR.

spinning side bands, which were not a problem at the muchlower magnetic field used in our study. The relative inten-sity of the 141-159 ppm region (area E) arises almost entirelyfrom the phenolic carbons C3 and C4 of the guaiacyl ligninunit (Fig. 2A), which predominates in softwoods. Therefore,the percentage of total C due to the sum of the four aromatic(C1, C2, C5, C6) and two phenolic (C 3 , C4 ) carbons oflignin monomer units can be calculated as 3E, and that dueto the three carbons of the lignin side chain (Ca , Co, CO,as 1.5E. The 50-60 ppm region (area B) arises largely fromthe single methoxyl C of guaiacyl units; thus total lignin Cis given by

lignin C = 4.5E + BThe 60-96 ppm region (area C) arises from C2 to C6 of

cellulose and hemicellulose monomer units (Fig. 2B), whichpredominate in spectra of fresh wood, as well as from thethree side-chain carbons of lignin. Including intensity dueto the anomeric C 1 , the total contribution of carbohydrateC is then calculated from

carbohydrate C = 1.2(C - 1.5E)When this calculation was done for the sample of highlydecomposed western red cedar (decay class IV'), the valueof 1.5E was greater than that of area C. Therefore, for thissample carbohydrate C was assumed to be zero, and thewhole region from 60 to 159 ppm (areas B-E) was attributedto lignin C.

The ratio of carbohydrate to lignin monomer units(CmLm) was calculated from

Cm 1.2(C - 1.5E) 13/

- 3E

An alternative calculation was made of aromatic ligninC (Ar*), by correcting area D for the intensity due to carbo-hydrate anomeric C:

(B) cellulose repeating unit

6CH2OH

H 5C —0I /1. r o C4—

E? ti- 0 \OH H/11

63--k

H OHFIG. 2. Repeating unit of (A) guaiacyl and (B) cellulose

structural units of softwood lignin.

[4J Ar* = D - 0.2(C - 1.5E)This value was used in the alternate calculation of the ratioof carbohydrate to lignin monomer units

Cm* 1.2(C - 1.5E)

To assess further the validity of analyzing the NMRspectra in this way, three other ratios were calculated fromNMR intensities attributed to lignin. Based on the structuralunit of guaiacyl lignin, the ratio of aromatic to phenoliclignin C should be 2; this is calculated from

aromatic C Ar*phenolic C E

The ratio of aromatic plus phenolic to methoxyl C shouldbe six, and this was calculated in two ways:

aromatic C + phenolic C3E=methoxyl C

[7b] aromatic C + phenolic C E Ar*

methoxyl CSome uncertainties are inherent in analyzing the NMR

spectra in this way. Intensity distributions in CPMAS spectramay be distorted (Alemany et al. 1983; Fyfe 1984), althoughthe conditions used in the present study have been shown

LmE + Ar*

[7a]

1385PRESTON ET AL.

TABLE 1. Selected physical and chemical properties, ratios of carbohydrate to lignin monomer units (Cm/L„,),and intensity ratios (as defined earlier) for the samples examined by 13C CPMAS NMR

Sample AT°C

(mg•g -I)N

(mg-g -I)Cra/Ln,(eq. 3)

Cm/Lm*(eq. 5)

Aromatic*,E

(eq. 6)

3E,OMe

(eq. 7a)

E + aromatic*,OMe

(eq. 7b)Douglas-fir

Fresh 3 464 0.3 3.22 2.89 2.34 4.22 4.71Class I 1 476 0.9 3.80 3.37 2.39 3.67 4.14Class II 1 473 1.0 3.74 3.57 2.15 4.55 4.77Class III 2 547 1.0 0.17 0.16 2.13 5.72 5.97Class IV 2 512 2.4 0.22 0.20 2.22 5.13 5.51Class VA 1 532 3.2 0.16 0.15 2.10 6.08 6.28Class Vs 1 499 4.7 0.30 0.26 2.49 5.97 6.96Class VR 1 537 3.8 0.025 0.025 1.97 6.33 6.26Klason lignin 577 1.5

Western hemlockFresh 2 462 0.2 2.22 2.21 2.02 4.76 4.79Class I 1 485 0.8 2.45 2.49 1.95 5.44 5.34Class II 2 479 1.1 1.69 1.70 1.98 5.14 5.10Class III 2 523 1.2 0.20 0.22 1.73 6.55 5.97Class IV 2 516 1.8 0.30 0.30 1.95 5.54 5.44Klason lignin 543 1.0

Western red cedarFresh 2 467 0.4 2.24 2.18 2.08 4.36 4.49Class I 1 487 1.1 2.05 1.87 2.29 4.27 4.68Class II 3 474 1.4 2.14 2.13 2.01 4.55 4.56Class III 3 471 1.7 2.07 2.04 2.04 4.54 4.60Class IV 2 473 2.1 1.71 1.60 2.20 5.00 5.34Class IV' 1 547 2.5 5.96 5.15Klason lignin 555 1.4

°Sample size.

to produce quantitatively reliable spectra for wood (Hatcher1988; Haw et al. 1984). Consistent with this, we found nosignificant difference in intensity distribution between spec-tra of Douglas-fir class V obtained using 1 vs. 2 ms cross-polarization time. Also, there are problems of peak overlapand lack of completely specific chemical shift regions, forwhich the use of vertical divisions and correction factors can-not fully compensate. Thus, it was not possible to separatehemicellulose from cellulose, and the analysis of ligninsignals was based only on the guaiacyl structural unit, whichis the major component of softwood lignin. Nonetheless,useful comparisons can be made of relative contributionsof different classes of C compounds for the different speciesand decay classes, and the internal checks on the validityof the procedure gave reasonable values, as discussed later.

As is often the case with NMR studies of this type, it wasnot possible to carry out many replicate measurements. Thiswas due mainly to time constraints, as each spectrumrequired several hours of acquisition time. For this reason,we first ran one spectrum of each sample type, and then fol-lowed up with replicates where these were considered mostessential. The number of samples run for each species anddecay class is given in Table 1. Where replicate spectra wereobtained, there was good agreement (within 5%) of therelative areas for samples from the same species and decayclass. The exception was western red cedar of decay class IV,where two samples showed little decomposition, but one(designated IV ' ) was highly decomposed. (It should bepointed out that the time constraints arose largely from hav-ing to run the spectra on borrowed time in a chemistrydepartment, a common situation for agroforestry research.There is in fact, no NMR instrument available for forestry

research in Canada, despite the heavy utilization of NMRin other applied areas.)

Results and discussionCharacterization of fresh heartwood

The chemical shift assignments of the spectra are basedon previous studies of wood (Barron et al. 1985; Hatcheret a!. 1981; Haw et al. 1984; Hemmingson and Newman1985; Kolodziejski et al. 1982), cellulose (Earl andVanderHart 1981; Maciel et al. 1982; VanderHart andAtalla 1984), and lignin (Barron et al. 1985; Hatcher 1987;Hatcher et a!. 1981; Hatfield et al. 1987; Leary et al. 1986).The spectra for fresh heartwood of the three species weresimilar. As illustrated for Douglas-fir (Fig. 1A), the spec-tra are dominated by signals due to cellulose in the60-110 ppm region. These include the crystalline (65 ppm)and noncrystalline (62 ppm) components of C6, the C2, C3,and C5 ring carbons (72 and 75 ppm), the crystalline(89 ppm) and noncrystalline (84 ppm) components of CA,and the anomeric C 1 (105 ppm). The splitting in the intenseC2 , 3 ,5 peak, with higher intensity for the component at72 ppm, is also diagnostic of a crystalline cellulose compo-nent. Weak signals for the acetyl methyl (22 ppm) andcarbonyl (173 ppm) groups of hemicellulose could beobserved for all three species, but were of lower intensityfor western red cedar. Other carbons of hemicellullose occurin the same chemical shift region as those of cellulose. Inparticular, hemicellulose contributes to the intensity at84 ppm, and also at 103 ppm, where it produces a slightshoulder on the signal for the anomeric C of cellulose at105 ppm.

200 150 100 50 0

Residence Time

(A)

Decay Class

192

II 9

50 0200 150 100

stt_.,_ 81

III 25

IV

4

rt.

1386 CAN. J. FOR. RES. VOL. 20, 1990

DOUGLAS-FIR

PPM•

FIG. 3. Stacked plots of 13C CPMAS NMR spectra ofDouglas-fir of decay classes I-V.

Signals from lignin are seen at 56 ppm for methoxyl C,and from 110 to 160 ppm for aromatic and phenolic C. Inthe phenolic region from 141 to 159 ppm, guaiacyl C3occurs at 148 ppm, while the signal from the guaiacyl C4includes free C4-OH at 146 ppm and C4 participating inCo-O-C4 ether linkages at 153 ppm (Leary et al. 1986). Thisresults in a broad phenolic signal with a slight shoulder at153 ppm. The poorly resolved aromatic region has tworather broad maxima. One occurs at 132 ppm and is assignedto guaiacyl C 1 . The second, assigned to C2, C3, and C6 ofguaiacyl units, occurs at 115 ppm for western red cedar andDouglas-fir. For western hemlock the maximum is closerto 120 ppm for fresh heartwood but at the normal shift of115 ppm for decayed samples. There is some uncertainty inthe detailed assignment of carbons in this region; thechemical shift of guaiacyl C2, Cs, and C6 is usuallyreported as 115 ppm, although Hatfield et a!. (1987)reported a lower field value for C6, 123-125 ppm.

Spectra from samples of fresh heartwood of the threespecies were generally similar to each other and to publishedspectra for softwoods, including spruce (Hatcher et al. 1981;Leary et al. 1986), lodgepole pine (Kolodziejski et al. 1982),radiata pine (Hemmingson and Newman 1985), hoop pine(Barron et a!. 1985), and redwood (Leary et a!. 1986). TheDouglas-fir heartwood differed from the other two speciesin having a higher ratio of carbohydrate to lignin C(Table 1).Effects of decomposition

The 13C CPMAS NMR spectra for samples of increas-ing decay class are shown in Fig. 3, 4, and 5 for Douglas-fir, western hemlock, and western red cedar, respectively.

WESTERN HEMLOCK

Decay Class Residence Time

(A)

FIG. 4. Stacked plots of 13C CPMAS NMR spectra of westernhemlock of decay classes I-IV.

The graphs in Fig. 6 illustrate the changes with increasingdecay class for the distribution of C of the followingchemical types: aliphatic (area A, 0-50 ppm); carboxyl andcarbonyl (areas F + G, 159-210 ppm); total lignin [1]; andtotal carbohydrate [2]. Ratios derived from areas ofchemical-shift regions [3, 5, 6, 7a, 7b] are shown in Table 1.

In general, the changes observed in the NMR spectra withincreasing decay class and residence time (Figs. 3-5) are con-sistent with loss of cellulose and hemicellulose and with con-centration of more resistant lignin, waxes, and resins. Therealso appears to be some oxidation, as indicated by smallincreases of relative intensity due to carbonyl C (Fig. 6).There are some small changes in the aromatic region of thelignin spectra, but as discussed later, these do not seem con-sistent with major structural alterations of lignin withdecomposition. For western hemlock and Douglas-fir ofdecay class III and higher, the broad region around 115 ppmshows some separation into incompletely resolved compo-nents at 115 and 122 ppm, with the intensity of the lattergenerally being higher for the more decomposed samples;this was also seen for the well-decomposed western red cedarsample IV' . The spectrum for Douglas-fir V R is similar toa previously published spectrum from a highly decayedDouglas-fir log, also from Mount Rainier (Hatcher 1987);it showed a shoulder at 122 ppm rather than a resolved max-imum. This change in the aromatic region may be due toreduction in structural heterogeneity as the nonlignin and

I-4

the more easily decomposable lignin units are removed,although further investigation of this point would be useful.

As found previously (Sollins et a!. 1987), western hemlockpassed through the stages operationally defined by the decayclasses more quickly than Douglas-fir. The changes in theorganic components, however, followed a similar patternfor both species (Figs. 6A, 6B). In the first stage, from freshheartwood to Douglas-fir class II and western hemlockclass I, there was a small increase in the concentration of

III

I050200 150 100

II

30

, .......... • ,,,,,,,,,,,,,,,,, , ,,,,,, • ,,,,,,,, • • 0

(B) DOUGLAS -FIR100

20

80-

60

40•

•

0.5

0.4

- 0.3

r 0.2

- 0.1• •

20

0

AGE(A) P 4 9 25 81

DECAY CLASS F I II III IV

0.1

PRESTON ET AL. 1387

PPMFIG. 5. Stacked plots of 13C CPMAS NMR spectra of western

red cedar of decay classes I, II, III, and IV ' .

carbohydrate C. This likely reflects an enhancement of thecellulose content, as hemicellulose is removed preferentiallyin the early stages of decay (Kirk and Highley 1973).Increases in cellulose content in the early stages of decayhave also been observed for logs of Abies amabalis (Hope1987) and Abies balsamea (Lambert et al. 1980).

In the second stage (Douglas-fir classes II and III andwestern hemlock classes there was extensive loss ofcarbohydrate, with increasing concentration of lignin C. Atthe same time, the relative intensities of aliphatic andcarbonyl C increased slightly. For alkyl C, this is probablydue to the selective preservation of waxes and resins foralkyl C, while oxidation contributes to the increase incarbonyl C. The third stage is represented by Douglas-firclasses III-Vs and western hemlock classes HI and IV.Changes in the spectra were small and inconsistent, withcarbohydrate content remaining at about 10% of total C.

Both methods of assessing the ratio of carbohydrate tolignin monomers [3 and 51 gave similar results (Table 1).For both species, C./ L,„ increased initially, then declinedto 0.2-0.3 for most samples of decay classes III-V. For theoldest Douglas-fir sample (V R), however, loss of cellulosehad advanced further, and Cm/Ln, was reduced to 0.025.Spectral features indicating a crystalline component ofcellulose, as described earlier, could be observed throughclass III for all species.

Most spectra for western red cedar showed little changefrom fresh heartwood to class IV, with Cm/Lm decreasingfrom 2.2 to 1.6 (Table 1). In contrast, physical appearance(Sollins et al. 1987) and density (Fig. 6C) of the western redcedar logs changed considerably over this sequence. Slowfungal colonization of western red cedar heartwood, relativeto Douglas-fir and western hemlock, may explain the lack

(A) WESTERN HEMLOCK

80 0.4

•60 0.3

40 02

1-1

•

DECAY CLASS F I II III IV VA VSv1s43

AGE(A) 0 3 11 50 87

192VR

(C) WESTERN RED CEDAR

. AN-- 0.5100:,.

80-' * -0.4

60- . - 0.3

40 - 0.220_ ...... ..... . "

- 0.1

0 'I., i0 5 17 30

o85AGE(A)

DECAY CLASS F III III IV(IV.)

FIG. 6. Changes in classes of organic carbon (as percentage oftotal spectral area) for increasing decay class and residence timefor (A) western hemlock, (B) Douglas-fir, and (C) western redcedar. The mean density for each decay class (right-hand axis) isalso shown (density data from Sollins et al. 1987). F, freshheartwood.

0 ,

of change in chemical properties. In a previous study,western red cedar was the only species that showed noevidence of fungal rhizomorphs at any stage of decay (Sollinset al. 1987). Western red cedar heartwood is also unusuallyresistant to bacterial and fungal growth because of the pres-ence of thujaplicins (Nault 1988).

The results obtained from one sample of the class IVwestern red cedar samples were exceptional. Designated aswestern red cedar IV' and shown separately in Figs. 5and 6C and in Table 1, it appeared to have lost all carbo-hydrate C, while the intensity from 60 to 96 ppm was lessthan 1.5E, insufficient to account for the three side-chaincarbons of the lignin monomer unit. The contrast in theNMR spectra between this and the other two class IVwestern red cedar samples is also consistent with the dif-ferences in C content (Table 1). Carbon content of westernred cedar samples showed little change from 467 mg • g -'for fresh heartwood to 473 mg • g I for decay class IV, but

WESTERN RED CEDAR

Residence Time'

Decay Class (A)

I V'

85

% OF TOTAL C

100DENSITY (g.cm-3)

0.5

Aliphatic C

Carbonyl C

Lignin C

Carbohydrate C

Density

DOUGLAS-FIR

(A) Class VR

DEPHASING DELAY(la)

1200 150 100

80

60

40

I 1 1200 150 100

PPM0

150

1388

CAN. J. FOR. RES. VOL. 20, 1990

FIG. 7. Dipolar-dephased 13C CPMAS NMR spectra withincreasing dephasing time for (A) Douglas-fir decay class VR and(B) Douglas-fir decay class I.

was 546 mg . g -1 for the IV' sample, closer to the valuesfound for classes III-V for the other two species.

The preferential loss of carbohydrate seen for Douglas-fir and western hemlock is typical of that due to brown-rotfungi (Jurgensen et al. 1987; Kirk and Highley 1973). How-ever, despite the role of insects and fungi in decomposition(Edmonds and Eglitis 1989; Harmon et a!. 1986; Jurgensen

et al. 1987; Kirk and Highley 1973; Seastedt and Tate 1981),there was no NMR evidence for accumulation of chitin(poly-N-acetyl-p-glucosamine) in any of the three speciesstudied. The chemical shifts of chitin are largely coincidentwith those of cellulose, hemicellulose, and the ligninmethoxyl (Preston et al. 1986; Saito et al. 1981). However,an accumulation of chitin during decomposition would beaccompanied by increasing intensity of the two acetyl signalsat 23 and 175 ppm (Preston et al. 1986). Lack of NMRevidence for chitin is consistent with reports that chitindecomposes within a few weeks in the soil (Gould et al. 1981;Okafor 1966), and also that the concentration of fungalmaterial in decayed wood, apart from portions wheremycelium is present, has been estimated to be quite low,about 2% (Cowling 1961 as cited by Kirk 1975). To bedetected by NMR in these samples, chitin would probablyhave to constitute 5% of total C. In addition to biologicalmechanisms, slow chemical autohydrolysis may also havecontributed to loss of carbohydrate (Hedges et al. 1985).

There was also no accumulation of aliphatic intensity at0-50 ppm, in contrast with that found with increasingdecomposition in peats (Hammond et al. 1985; Orem andHatcher 1987a; Preston et al. 1987, 1989) and organichorizons of forest soils (Hempfling et al. 1987; Wilson et a!.1983; Zech et al. 1987). The broad weak signal observed inthe 0-50 ppm region in some of the more decomposedsamples is most likely due to recalcitrant resins and waxesfrom the original wood (Hatcher 1987, 1988; Hatcher et al.1981).

Investigation of lignin structureThree approaches were used to assess whether structural

alterations of lignin had occurred during decomposition.First, examination of the three intensity ratios in Table 1(from eqs. 6, 7a, and 7b) suggested that lignin had persistedwith little structural alteration. The ratio of aromatic tophenolic C [6] remained close to 2 for all decay classes andspecies. The ratio of aromatic plus phenolic C to methoxyl C[7a, 7b] should be 6. In most cases the ratio was less than6, but tended to increase with increasing decomposition toapproximately 5.5-6. The ratios found for the more decayedsamples probably reflect more accurately the lignin struc-ture; measurements of methoxyl C intensity in the lessdecomposed samples would tend to be exaggerated becausethe peak is not resolved from the region of high carbo-hydrate intensity and also because there would be some con-tribution from hemicellulose methoxyl. These interferenceswould tend to lower the ratio; but with progressive removalof hemicellulose and cellulose, the ratios generallyapproach 6, indicating little change in the basic guaiacylstructural unit with decomposition.

Second, samples representing the most advanced statesof decomposition of the three species (Douglas-fir class VR,western hemlock class IV, and western red cedar class IV')were hydrolysed with H2504, corresponding to the laststage of isolation of Mason lignin. Figure 1C shows the spec-trum obtained for Klason lignin from the Douglas-firsample; those from the western hemlock and western redcedar samples were similar. The spectra were similar topublished spectra of Klason lignin prepared from fresh wood(Cyr et a!. 1988; Haw et al. 1984; Kolodziejski et a!. 1982;Leary et al. 1986) and indicate that the hydrolysis removedall of the residual cellulose. Hydrolysis causes other changes

PRESTON ET AL. 1389

in the spectra; there was an increase of the relative intensityat 0-50 ppm, while the aromatic region showed broaden-ing and loss of intensity of the 132 ppm peak, which wasreduced to a shoulder of the peak at 125 ppm. Apart fromremoval of cellulose, chemical changes caused by H2SO4hydrolysis are not well understood, but are most likely dueto some de-etherification at C4 (Leary et al. 1986) and con-densation reactions that form C—C bonds (Cyr et al. 1988).Incidental to this preparation of Klason lignin, we obtainedvalues for the proportion of N hydrolysable by H2SO4. Thepercentage of N lost upon hydrolysis was 70% for Douglas-fir, 67% for western hemlock, and 60% for western redcedar.

Finally, a sample of Douglas-fir VR was examined usingdipolar dephasing (Fig. 7A) with a series of increasingdephasing times (Opella and Frey 1979; Hatcher 1987, 1988).In this pulse sequence, intensity is lost more quickly fromcarbons that have strong dipolar interactions with protons.The dipolar interaction is weakened in two cases: for non-protonated C by the longer separation from hydrogen nuclei,and for methyl C by methyl group rotation, which occurseven in solids (Fyfe 1984; Opella and Frey 1979). The dipolardephased spectra in Fig. 7A show features consistent withunaltered lignin, namely peaks for methoxyl at 56 ppm,phenolic at 148 ppm with a shoulder at 153 ppm, and non-protonated aromatic C (guaiacyl C 1 ) at 132 ppm. Only theweaker carboxyl (172 ppm) and carbonyl (195 ppm) peaksindicate that some oxidation has occurred.

Thus there appeared to be little structural alteration ofthe remaining heartwood lignin during decay. The NMRspectra showed slight evidence of oxidation, and thereappeared to be some loss from the three-carbon side chainfor one well-decomposed sample of western red cedar (IV' ).This contrasts with the structural alterations of lignin thathave been found in incubation studies of wood with white-rot fungi, and to a lesser extent with brown-rot fungi (Enokiet al. 1988; Kirk 1975; Kirk and Chang 1975; Nilsson et al.1989), and with the indications of lignin degradation andhumification in litter and soil (Hatcher et al. 1989;Hempfling et al. 1987; KOgel 1986).

The persistence of lignin in these large logs may be duein part to the greater resistance to decay of guaiacyl-basedlignin than the syringyl-based lignin of angiosperms (Hedgeset al. 1985; 1988; Nilsson et a!. 1989). In addition, moisturemay be inhibiting lignin decay. With increasing residencetime on the forest floor, the moisture content of the logsincreased to approximately 350% in winter and 250% insummer for decay class IV (Sollins et al. 1987). Fungaldecomposition of ligriin, which is primarily an oxidative pro-cess, should be restricted if conditions became anaerobic dueto waterlogging (Hedges et al. 1985, 1988; Kirk 1975; Kirkand Chang 1975; Orem and Hatcher 1987b). Limitation ofsunlight at the forest floor might also restrict the potentialfor photodegradation of lignin (Hemmingson and Morgan1989). Nutrient limitations, especially of N (Sollins et al.1987; Jurgensen et a!. 1987), and lack of mechanisms forfragmentation and mixing with the soil may also limitdecomposition.

Figure 7B illustrates complications that arise with the useof dipolar dephasing for a sample with high cellulose con-tent, Douglas-fir class 1. Unlike the spectra for the well-decomposed Douglas-fir class VR and previously reporteddipolar-dephased spectra of lignin (Hatcher 1987), the inten-

sity due to CH and CH2 carbons does not decay to zeroafter 40 ps of dephasing time, and oscillatory behaviour isobserved for the cellulose peak at 72 ppm. A similar oscilla-tory behaviour was seen for spectra taken with a constantdephasing time of 60 ps and spinning speeds varying from2000 to 5000 Hz (spectra not shown). This phenomenon canoccur when dipolar interactions are weakened by molecularmotion and can also be caused by the magic-angle spinningitself when the spinning is done at high speeds (Alemanyet al. 1983; Newman 1990).

ConclusionsSolid-state 13C NMR can elucidate patterns of decom-

position in the organic components of woody litter decay-ing on the forest floor. For Douglas-fir and western hemlocklogs, the major process appears to be loss of cellulose, withconcomitant concentration of the more resistant lignin andresins, and a small amount of oxidation. The process wasnearly twice as fast in western hemlock as in Douglas-fir.The pattern of decomposition for western red cedar wascompletely different; the NMR spectra generally indicatedlittle change in the relative abundance of organic compo-nents despite loss of density and other indicators of decom-position. Thus, samples of western red cedar examined inthis study showed little evidence of chemical change, despitemass loss and physical collapse in the older logs. For all threespecies, there appeared to be little degradation of ligninstructures. Throughout the decay series, the NMR spectracan be interpreted for the most part by changes in theproportions of the original components in the wood. Therewas no evidence for significant accumulations of chitin,humic substances, or aliphatic products of microbial activ-ity. The decay processes were consistent with activity byinsects, brown-rot fungi, and chemical autohydrolysis, withfurther decomposition of the highly ligneous residue appar-ently restricted by unfavourable environmental conditions.

The aims of this investigation were essentially qualitative,i.e., to attempt to elucidate in broad terms the changes inorganic components during decomposition, and to evaluatethe 13C CPMAS NMR technique for studies of decomposi-tion in large woody litter. The information provided byNMR can be further used to select the most appropriatechemical analyses of organic components, or to designfurther studies to answer the questions raised in this pre-liminary survey. In general, it should also be possible toapply the information available from NMR analysis todevelop a better understanding of the chemical and biolog-ical mechanisms controlling decay and nutrient release inwoody litter.

Acknowledgements

We thank A. Rusk, Pacific Forestry Centre, Victoria,British Columbia, for carrying out the Kjeldahl N analyses,and for assistance in preparation of the manuscript. We alsothank Ann Van Niekerk, Pacific Forestry Centre, for theC analyses.

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